It is known that extracellular production of desired foreign proteins in E. coli is a very efficient method in the sense that: the secreted foreign proteins are protected against proteolysis by proteolytic enzymes in E. coli, the secretion process guides appropriate folding of foreign proteins to inhibit the formation of insoluble inclusion bodies, and the N-terminal secretion signal peptide is removed from foreign proteins during the secretion process to keep the amino acid sequence identical to the naturally occurring one. This method also allows mass production of foreign proteins through high concentration culture and continuous culture. Furthermore, this method makes for pure purification of foreign proteins because little bacterial proteins are secreted into culture media.
Since the extracellular production has several advantages as mentioned above, various studies on the extracellular production systems have been actively pursued to produce desired foreign proteins in E. coli. The extracellular production systems developed so far are classified in the following three categories: the first one is a method for extracellular production by the recombination of a secretion signal sequence and a desired foreign protein. For example, Toksoy et al. produced TaqI protein on the cell surface employing a fusion protein containing a secretion signal sequence and maltose binding protein(MBP), Lo et al. produced β-1,4-endoglucanase of Bacillus subtilis on the cell surface of E. coli, and Nagahari et al. produced β-endorphine on the cell surface of E. coli through the recombination of the OmpF secretion signal peptide and 8 amino acids from the N-terminus of OmpF. In addition, Yamamoto et al. tried to produce p21 protein from harvey murine sarcoma virus extracellularly by using the OmpF secretion signal sequence. However, it turned out that p21 was not produced on the cell surface, but accumulated in inclusion bodies(see: Toksoy E. et al., Biotechnology Techniques, 13:803-808, 1999; Lo A. C. et al., Appl. Environ. Micrbiol., 54:2287-2292, 1988; Nagahari et al., EMBO J., 4:3589-3592, 1985; and, Yamamoto et al., Appl. Micobiol. Biotechnol., 35:615-621, 1991).
The second classification is a method for extracellular production by the recombination of a secretion protein from E. coli and a desired protein. For example, Baneyx et al. produced OmpA-TEM-β-lactamase fusion protein on the cell surface together with TolAIII membrane protein of E. coli, Robbens et al. used kil gene to produce interleukin-2, van der Wal et al. used a lipoprotein, BRP(bacteriocin release protein), to produce β-lactamase on the cell surface, and Aristidou et al. increased the yield of extracellular production using BRP by addition of glycine to culture media(see: Baneyx F. and Eugene W. M., Protein Expr. Purif., 14:13-22, 1998; Robbens J. et al., Protein Expr. Purif., 6:481-486, 1995; van der Wal F. J. et al., Appl. Environ. Microbiol., 64:392-398, 1998; and, Aristidou A. A. et al., Biotechnol. Lett., 15:331-336, 1993).
The third classification is a method for extracellular production by the aid of an outer membrane-free E. coli, such as an L-type strain of E. coli, a mutant that has only an inner cellular membrane without an outer cellular membrane and periplasm. The extracellular production of foreign proteins is simpler than prior methods in the culture of an L-type strain because expressed proteins are transported through only the inner cellular membrane to be secreted into culture media. For example, Kujau et al. used RV308 strain, an L-type mutant, to produce a miniantibody(miniAb) on the cell surface(see: Kajau M. J. et al., Appl. Microbiol. Biotechnol., 49:51-58, 1998).
As expounded above, a variety of methods have been developed to produce desired foreign proteins on the cell surface of E. coli. Most of these prior art methods are, however, proven to be less satisfactory because partial degradation of some foreign proteins by bacterial proteolytic enzymes makes the purification process complex and makes high concentration cell culture impossible. In addition, extracellular production employing an L-type strain of E. coli has shortcomings in that the said strain is not suitable for high concentration cell culture due to its weak resistance to environmental stress and its short life cycle.
Under the circumstances, there are strong reasons for exploring and developing an alternative method for extracellular production of desired foreign proteins on the cell surface of E. coli. 